Abstract Corneal endothelial (CE) cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. A new procedure, Descemet's stripping automated endothelial keratoplasty (DSAEK), which is superior to the traditional technique in many aspects, requires extensive manipulation of the corneal endothelium, resulting in extensive CE cell loss. Thus, future development of late CE failure in eyes that have received DSAEK is likely. Previously, We have reported how a CE autocrine trophic factor, the 28 amino-acid vasoactive intestinal peptide (VIP) maintains the differentiated state of the corneal endothelium, including CE cell size, shape, and retention, in situ in the human donor cornea, whereas exogenous VIP protects it against the killing effect of oxidative stress. VIP increases synthesis of the CE cell differentiation marker N-cadherin, the cell-cell adhesion molecule, and that of the anti-apoptotic protein Bcl-2. The endogenous VIP in CE cells is upregulated by ciliary neurotrophic factor (CNTF), which is also a CE autocrine trophic factor released by CE cells surviving the oxidative stress. The CNTF receptor (CNTFR) is expressed in CE cells in human donor cornea and gradually becomes lost during corneal storage. By applying this knowledge gained from studying CE cell physiology to the enhancement of CE cell survival in corneal transplantation, which is our goal, we will also validate the relevance of this knowledge. Our specific aims are to demonstrate that 1. VIP treatment prior to storage of freshly dissected human donor corneas increases their CE cell N-cadherin, Bcl-2, CNTFR, and retention levels in corneal storage, 2. One additional VIP treatment at the time of microkeratome cutting of the human donor corneas stored for DSAEK brings about beneficial effects described in aim 1 and decreased CE cell apoptosis and injury to precut corneas for DSAEK, 3. VIP treatments prior to storage of freshly dissected human donor corneas and prior to microkeratome cutting produce superior precut corneas for DSAEK demonstrating decreased CE damage in an established in vitro endothelial keratoplasty model, 4. Intra-operative VIP (or CNTF) treatment decreases CE damage in an established in vitro endothelial keratoplasty model.